Potent Protein Kinase C Activator Research Articles

Synthesis and Biological Activities of Simplified Analogs of the NaturalPKCLigands, Bryostatin‐1 and Aplysiatoxin

Protein kinase C (PKC) isozymes play central roles in signal transduction on the cell surface and could serve as promising therapeutic targets of intractable diseases like cancer, Alzheimer's disease, and acquired immunodeficiency syndrome (AIDS). Although natural PKC ligands like phorbol esters, ingenol esters, and teleocidins have the potential to become therapeutic leads, most of them are potent tumor promoters in mouse skin. By contrast, bryostatin-1 (bryo-1) isolated from marine bryozoan is a potent PKC activator with little tumor-promoting activity. Numerous investigations have suggested bryo-1 to be a promising therapeutic candidate for the above intractable diseases. However, there is a supply problem of bryo-1 both from natural sources and by organic synthesis. Recent approaches on the synthesis of bryo-1 have focused on its simplification, without decreasing the ability to activate PKC isozymes, to develop new medicinal leads. Another approach is to use the skeleton of natural PKC ligands to develop bryo-1 surrogates. We have recently identified 10-methyl-aplog-1 (26), a simplified analog of tumor-promoting aplysiatoxin (ATX), as a possible therapeutic lead for cancer. This review summarizes recent investigations on the simplification of natural PKC ligands, bryo-1 and ATX, to develop potential medicinal leads.

Transcriptional suppression of human apolipoproteina4 and apolipoproteinc3 genes by phorbol myristate acetate in hepatic and intestinal cells

Genetic, epidemiological and clinical evidence has demonstrated the importance of the human apolipoproteinA5 (apoA5), apolipoproteinA4 (apoA4), apolipoproteinC3 (apoC3), and apolipoproteinA1 (apoA1) genes in the control of the triglyceride and cholesterol concentrations in the blood. However, little is known about the mechanism by which protein kinase C (PKC) regulates the expression of these genes in hepatic and intestinal cells. The aim of this study was to explore the regulatory role of PKC on the expression of apoA5, apoA4, apoC3 and apoA1. Hepatic HepG2 and intestinal Caco-2 cells were treated with a potent PKC activator, Phorbol myristate acetate (PMA). The real time quantitative RT-PCR (qRT-PCR) technique was used to evaluate the effects of PMA on the expression of apoA1, apoA4, apoA5 and apoC3 genes. Nuclear run on assay was used to determine whether the effect of PMA on apoA4 and apoC3 was due to its ability to regulate the transcription of these genes. PMA specifically down-regulated the transcription of apoA4 and apoC3, but exhibited no effects on apoA1 or apoA5 in either HepG2 or Caco-2 cells. Further study by nuclear run on assay proved that the suppressive effect of PMA on apoA4 and apoC3 resulted from PMA's regulation of the transcription rate of the two genes. PMA down-regulated transcription of apoA4 and apoC3 possibly through the common regulatory element shared by these two genes, suggesting a suppressive role of PKC on the transcriptional regulation of specific apolipoproteins in hepatic and intestinal cells.

Oxygen-sensitive reduction in Ca2+-activated K+ channel open probability in turtle cerebrocortex

In response to low ambient oxygen levels the western painted turtle brain undergoes a large depression in metabolic rate which includes a decrease in neuronal action potential frequency. This involves the arrest of N-methyl-d-aspartate receptor (NMDAR) and α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor (AMPAR) currents and paradoxically an increase in γ-aminobutyric acid receptor (GABAR) currents in turtle cortical neurons. In a search for other oxygen-sensitive channels we discovered a Ca2+-activated K+ channel (KCa) that exhibited a decrease in open time in response to anoxia. Single-channel recordings of KCa activity were obtained in cell-attached and excised inside-out patch configurations from neurons in cortical brain sheets bathed in either normoxic or anoxic artificial cerebrospinal fluid (aCSF). The channel has a slope conductance of 223pS, is activated in response to membrane depolarization, and is controlled in a reversible manner by free [Ca2+] at the intracellular membrane surface. In the excised patch configuration anoxia had no effect on KCa channel open probability (Popen); however, in cell-attached mode, there was a reversible fivefold reduction in Popen (from 0.5±0.05 to 0.1±0.03) in response to 30-min anoxia. The inclusion of the potent protein kinase C (PKC) inhibitor chelerythrine prevented the anoxia-mediated decrease in Popen while drip application of a phorbol ester PKC activator decreased Popen during normoxia (from normoxic 0.4±0.05 to phorbol-12-myristate-13-acetate (PMA) 0.1±0.02). Anoxia results in a slight depolarization of turtle pyramidal neurons (∼8mV) and an increase in cytosolic [Ca2+]; therefore, KCa arrest is likely important to prevent Ca2+ activation during anoxia and to reduce the energetic cost of maintaining ion gradients. We conclude that turtle pyramidal cell Ca2+-activated K+ channels are oxygen-sensitive channels regulated by cytosolic factors and are likely the reptilian analog of the mammalian large conductance Ca2+-activated K+ channels (BK channels).

Novel regulation of protein kinase C-η

Protein kinase C (PKC) is the receptor for tumor promoting phorbol esters, which are potent activators of conventional and novel PKCs, but persistent treatment with phorbol esters leads to downregulation of these PKCs. However, PKCη, a novel PKC isozyme, resists downregulation by tumor-promoting phorbol esters, but little is known about how PKCη level is regulated. Phosphorylation and dephosphorylation play an important role in regulating activity and stability of PKCs. In the present study, we have investigated the molecular mechanism of PKCη regulation. Several PKC activators, including phorbol 12,13-dibutyrate, 12-O-tetradecanoylphorbol-13-acetate and indolactam V caused upregulation of PKCη, whereas the general PKC inhibitor Gö 6983, but not the conventional PKC inhibitor Gö 6976 led to the downregulation of PKCη. Upregulation of PKCη was associated with an increase in phosphorylation of PKCη. Silencing of phosphoinositide-dependent kinase-1, which phosphorylates PKCη at the activation loop, failed to prevent PKC activator-induced upregulation of PKCη. Knockdown of PKCε but not PKCα inhibited PKC activator-induced upregulation of PKCη. Thus, our results suggest that the regulation of PKCη is unique and PKCε is required for the PKC activator-induced upregulation of PKCη.

Immunoregulatory potential of marine algal toxins yessotoxin and okadaic acid in mouse T lymphocyte cell line EL-4

We have studied the effects of the marine algal toxins yessotoxin (YTX) and okadaic acid (OA) on the T cell receptor complex (TCR) expression, an important mechanism by which T cell responsiveness is controlled. Immune system cells are relevant targets to study the immunoregulatory potential of marine toxins since the immune system has been reported as one of the targets of marine algal toxins. This study reports results from exposing the mouse T lymphocyte cell line EL-4 to increasing concentrations of YTX and OA for 72h. We found that both YTX and OA affected TCR recycling kinetics and induced a specific and reversible TCR down-regulation in T lymphocyte EL-4 cells that was time and concentration dependent. Experiments using the potent protein kinase C (PKC) inhibitor stausporine indicated that YTX-induced TCR down-regulation was partially mediated by PKC activation. In contrast, OA-induced TCR down-regulation was mediated by the serine/threonine protein phophatase 2A (PP2A) inhibition. In summary, the results suggest that OA and YTX concentrations in a similar range than those detected in mice bloodstream after oral administration have the potential to adjust the T cell responsiveness during the initiation of T cell activation by affecting the TCR expression levels via PKC and PP2A activities.

The Relaxing Effect of α-Defensin 1 on the Adrenergic Responses of Rat Bladder

Defensins, cysteine-rich cationic polypeptides released from neutrophils, are known to have powerful antimicrobial properties. In this study, we sacrificed 30 rats to investigate the effects of α-defensin 1 on detrusor muscle contractions in isolated rat bladder. From the experiments we found relaxing effects of α-defensin 1 on the contractions induced by phenylephrine (PE) but not by bethanechol (BCh) in the detrusor smooth muscles. To determine the mechanisms of the effects of α-defensin 1, the changes of effects on PE-induced contraction by α-defensin 1 pretreatment were observed after pretreatment of Rho kinase inhibitor (Y-27632), protein kinase C (PKC) inhibitor (Calphostin C), potent activator of PKC (PDBu; phorbol 12,13-dibutyrate), and NF-κB inhibitors (PDTC; pyrrolidinedithiocarbamate and sulfasalazine). The contractile responses of PE (10(-9)~10(-4) M) were significantly decreased in some concentrations of α-defensin 1 (5×10(-9) and 5×10(-8) M). When strips were pretreated with NF-κB inhibitors (PDTC and sulfasalazine; 10(-7)~10(-6) M), the relaxing responses by α-defensin 1 pretreatment were disappeared. The present study demonstrated that α-defensin 1 has relaxing effects on the contractions of rat detrusor muscles, through NF-κB pathway. Further studies in vivo are required to clarify whether α-defensin 1 might be clinically related with bladder dysfunction by inflammation process.

Positive inotropic, positive chronotropic and coronary vasodilatory effects of rat amylin: mechanisms of amylin-induced positive inotropy

Even though there are a few studies dealing with the cardiac effects of amylin, the mechanisms of amylin-induced positive inotropy are not known well. Therefore, we investigated the possible signaling pathways underlying the amylin-induced positive inotropy and compared the cardiac effects of rat amylin (rAmylin) and human amylin (hAmylin).Isolated rat hearts were perfused under constant flow condition and rAmylin or hAmylin was infused to the hearts. Coronary perfusion pressure, heart rate, left ventricular developed pressure and the maximum rate of increase of left ventricular pressure (+dP/dtmax) and the maximum rate of pressure decrease of left ventricle (-dP/dtmin) were measured.rAmylin at concentrations of 1, 10 or 100 nM markedly decreased coronary perfusion pressure, but increased heart rate, left ventricular developed pressure, +dP/dtmax and -dP/dtmin. The infusion of H-89 (50 μM), a protein kinase A (PKA) inhibitor did not change the rAmylin (100 nM)-induced positive inotropic effect. Both diltiazem (1 μM), an L-type Ca2+ channel blocker and ryanodine (10 nM), a sarcoplasmic reticulum (SR) Ca2+ release channel opener completely suppressed the rAmylin-induced positive inotropic effect, but staurosporine (100 nM), a potent protein kinase C (PKC) inhibitor suppressed it partially. hAmylin (1, 10 and 100 nM) had no significant effect on coronary perfusion pressure, heart rate and developed pressure, +dP/dtmax and -dP/dtmin.We concluded that rAmylin might have been produced vasodilatory, positive chronotropic and positive inotropic effects on rat hearts. Ca2+ entry via L-type Ca2+ channels, activation of PKC and Ca2+ release from SR through ryanodine-sensitive Ca2+ channels may be involved in this positive inotropic effect. hAmylin may not produce any significant effect on perfusion pressure, heart rate and contractility in isolated, perfused rat hearts.

Receptor‐induced phasic activity of newborn mouse bladders is inhibited by protein kinase C and involves T‐type Ca2+ channels

To investigate the mechanisms involved in the phasic contractile activity after muscarinic receptor activation of newborn urinary bladders and to compare neonatal and adult bladders. Detrusor muscle strips were isolated from newborn mice (aged 0-2 days) and compared with preparations from adult mice (aged 10-12 weeks). The effects of an activator (phorbol 12,13-dibutyrate, PDBu) and an inhibitor (GF109203X) of protein kinase C (PKC) on contractions were investigated. T-type Ca2+ channels were blocked with NiCl2. The newborn urinary bladders responded with prominent phasic contractile activity in response to carbachol (1 microM). GF109203X (3 microM) reduced carbachol-induced force by approximately 60% in the newborn, compared with 30% in the adult. PDBu (1 microM) enhanced the muscarinic receptor-mediated contraction in adult bladder muscle, whereas it completely abolished the responses in the newborn. There was no inhibition after activation with depolarization (high-K+) or purinergic agonists (ATP, alpha,beta-methylene ATP). NiCl2 (>30 microM) inhibited the peak responses to carbachol in the newborn and at 300 microm it completely abolished the phasic contractile response. The responses of the adult bladder muscle were only marginally affected by NiCl2. Muscarinic receptor stimulation recruits the PKC signalling pathway in both the adult and neonatal urinary bladder. Potent PKC activation is inhibitory on carbachol-induced activation in the newborn and stimulatory in the adult bladder. Furthermore, muscarinic receptor stimulation activates T-type Ca2+ channels in the newborn, but not the adult bladder.

Involvement of Acid β-Glucosidase 1 in the Salvage Pathway of Ceramide Formation

Activation of protein kinase C (PKC) promotes the salvage pathway of ceramide formation, and acid sphingomyelinase has been implicated, in part, in providing substrate for this pathway (Zeidan, Y. H., and Hannun, Y. A. (2007) J. Biol. Chem. 282, 11549-11561). In the present study, we examined whether acid beta-glucosidase 1 (GBA1), which hydrolyzes glucosylceramide to form lysosomal ceramide, was involved in PKC-regulated formation of ceramide from recycled sphingosine. Glucosylceramide levels declined after treatment of MCF-7 cells with a potent PKC activator, phorbol 12-myristate 13-acetate (PMA). Silencing GBA1 by small interfering RNAs significantly attenuated acid glucocerebrosidase activity and decreased PMA-induced formation of ceramide by 50%. Silencing GBA1 blocked PMA-induced degradation of glucosylceramide and generation of sphingosine, the source for ceramide biosynthesis. Reciprocally, forced expression of GBA1 increased ceramide levels. These observations indicate that GBA1 activation can generate the source (sphingosine) for PMA-induced formation of ceramide through the salvage pathway. Next, the role of PKCdelta, a direct effector of PMA, in the formation of ceramide was determined. By attenuating expression of PKCdelta, cells failed to trigger PMA-induced alterations in levels of ceramide, sphingomyelin, and glucosylceramide. Thus, PKCdelta activation is suggested to stimulate the degradation of both sphingomyelin and glucosylceramide leading to the salvage pathway of ceramide formation. Collectively, GBA1 is identified as a novel source of regulated formation of ceramide, and PKCdelta is an upstream regulator of this pathway.

Mouse pigmentation mutants help identify a uveal melanoma oncogene

Mouse pigmentation mutants help identify a uveal melanoma oncogene

HTLV-1 Tax-induced NF-κB activation is synergistically enhanced by 12-O-tetradecanoylphorbol-13-acetate: mechanism and implications for Tax oncogenicity

Nuclear factor kappa B (NF-kappaB) factors regulate a wide range of physiological and oncogenic processes. Normally, these factors are transiently activated by specific external signals which induce their dissociation from inhibitors of kappaB (IkappaB) and subsequent translocation to the nucleus where p65 links to the cyclic adenosine monophosphate response element binding protein (CBP)-p300 and P/CAF coactivators that are essential for its transcriptional activity. The pathogenic potential of human T-cell leukemia virus type 1 (HTLV-1) Tax protein is partly ascribed to its capacity to constitutively activate NF-kappaB factors because constitutive activity of these factors play an important role in the pathophysiology of adult T-cell leukemia (ATL) and tropical spastic paraparesis-HTLV-1 associated myelopathy (TSP-HAM). In assessing the possibility of modulating Tax pathogenic potential by external factors, we focused here on 12-O -tetradecanoylphorbol-13-acetate (TPA) which is a potent protein kinase C (PKC) activator. There are conflicting reports regarding the effect of TPA and PKC on NF-kappaB. Therefore, we reassessed this issue and also investigated their influence on Tax-mediated activation of these factors. We found that TPA promoted NF-kappaB nuclear translocation and the DNA binding of p65 dimers through PKC activation. However, both TPA and ectopically expressed PKC had only a marginal effect on the transcriptional competence of these dimers, indicating that the DNA binding of such dimers is insufficient by itself for gene activation. Notably, however, both TPA and the ectopic PKC displayed strong synergistic enhancement of the Tax-induced activation of the NF-kappaB transcriptional function. In contrast, TPA and the ectopic PKC only slightly elevated the low activation of the NF-kappaB transcriptional capacity by cytoplasmic Tax mutants, indicating that the nuclear translocation of Tax was essential for this synergism. Subsequent experiments suggested that TPA contributed to this synergism by increasing the pool of free p65 which Tax could link to CBP and elevate, thereby, the amount of a p65-Tax-CBP ternary complex that could bind to NF-kappaB-responsive promoters and stimulate their expression. Finally, we demonstrated that this synergism operated also in HTLV-1-infected human T-cells. Earlier reports have shown a close linkage of pathological PKC-activating conditions (e.g., infectious and inflammatory diseases) to certain malignancies. On this ground, the present study suggests that such conditions may enhance the risk for ATL and TSP-HAM in HTLV-1 carriers by increasing the Tax-induced NF-kappaB activation.

Kinetic Analysis of the Interaction of the C1 Domain of Protein Kinase C with Lipid Membranes by Stopped-flow Spectroscopy

The diacylglycerol (DG)/phorbol ester-dependent translocation of conventional protein kinase C (PKC) isozymes is mediated by the C1 domain, a membrane-targeting module that also selectively binds phosphatidylserine (PS). Using stopped-flow spectroscopy, we dissect the contribution of DG/phorbol esters (C1 ligand) and PS in driving the association and dissociation of the C1 domain from membranes. Specifically, we examine the binding to membranes of the C1B domain of PKCbeta with a substituted Trp (Y123W) whose fluorescence is quenched upon binding to membranes. Binding of this construct (C1Bbeta-Y123W) to phospholipid vesicles is cooperative with respect to PS content and dependent on C1 ligand, as previously characterized. Stopped-flow analysis reveals that the apparent association rate (k(on)(app)), but not the apparent dissociation rate (k(off)(app)), is highly sensitive to PS content: the 60-fold increase in membrane affinity for vesicles containing no PS compared with 40 mol % PS results primarily from a robust (30-fold) increase in k(on)(app) with little effect (2-fold) on k(off)(app). Membrane affinity is also controlled by the content and structure of the C1 ligand. In contrast to PS, these ligands markedly alter k(off)(app) with smaller effects on k(on)(app). We also show that the affinity for phorbol ester-containing membranes is 2 orders of magnitude higher than that for DG-containing membranes primarily resulting from differences in k(off)(app). Our data are consistent with a model in which the C1 domain is recruited to the membrane via an initial weak electrostatic interaction with PS, followed by a rapid two-dimensional search for ligand, the binding of which retains the domain at the membrane. Thus, PS drives the initial encounter, and DG/phorbol esters retain the domain on membranes. The decreased effectiveness of DG compared with phorbol esters in retaining the C1 domain on membranes contributes to the molecular dichotomy of the rapid, transient nature of DG-dependent PKC signaling versus the chronic hyperactivity of phorbol ester-activated PKC.

Synergistic effects of chronic bryostatin-1 and α-tocopherol on spatial learning and memory in rats

Evidence is emerging that protein kinase C (PKC) plays a crucial role in the neural processing of memory information and that PKC deficits underlie certain types of memory impairment, including Alzheimer's dementia. Chronic activation of PKC isozymes with bryostatin-1 induces synthesis of the proteins that are involved in memory consolidation and, therefore, may represent a pharmacological strategy for antidementic and memory therapies. PKC isozymes are, however, sensitive to oxidants, whose generation is also increased by PKC activation. Oxidants may be responsible for some adverse effects with PKC activators, potentially limiting their antidementic and memory-enhancing “benefit”. We investigated the effects of intravenous bryostatin-1, a potent PKC activator, and of its co-administration with oral α-tocopherol, a potent antioxidant, on spatial learning and memory. Bryostatin-1 at a chronic and intravenous dose of 10 μg/m2 (2 doses/week for 3 weeks) alone did not significantly affect the spatial learning and memory, but showed a synergistic effect when co-administered with α-tocopherol (60 IU/kg, orally and daily for 3 weeks), a potent lipid-soluble antioxidant and also a possible inhibitor of PKC in peripheral tissues. Acute administration of the same doses, however, did not have obvious influence on the learning and memory. These results provide support for the strategy of achieving memory-enhancing benefits with PKC activators and restricting their oxidant-related adverse effects with α-tocopherol co-administration. These agents, therefore, may hold significant potential as new, combined antidementic and memory therapeutics in the future.

Angiotensin-Converting Enzyme Inhibitor Attenuates Monocyte Adhesion to Vascular Endothelium through Modulation of Intracellular Zinc

To elucidate an anti-inflammatory role of angiotensin-converting enzyme inhibitors (ACEIs) in cardiovascular disease, we studied the effect of ACEIs in monocyte adhesion to endothelial cells and underlying molecular mechanisms. Treatment of human monocytic THP-1 cells with monocyte chemoattractant protein-1 (MCP-1; 100 ng/ml; 10 min) significantly increased their adhesion to human umbilical vein endothelial cells (HUVECs) under flow condition (P < 0.001). Preincubation of THP-1 cells with imidaprilat (50 nM; 4 h), an active metabolite of imidapril, reduced MCP-1-triggered THP-1 cell adhesion (P < 0.01). Similar effects were obtained with experiments using human peripheral monocytes (P < 0.05). MCP-1 activated protein kinase C (PKC)alpha in THP-1 cells, resulting in the up-regulation of alpha4 and beta2 integrin. Imidaprilat attenuated MCP-1-induced PKC activation and integrin up-regulation in THP-1 cells. Imidaprilat also inhibited THP-1 cell adhesion induced by phorbol 12-myristate 13-acetate (PMA), a potent PKC activator. In attempt to elucidate the mechanisms for the modulation of PKC activity by imidaprilat, we found that MCP-1 or PMA increased labile zinc in THP-1 cells, which was canceled by imidaprilat. Indeed, zinc/pyrithione activated PKC and increased THP-1 cell adhesion. Zinc chelator as well as PKC inhibitor inhibited these processes, suggesting the role for labile zinc in PKC activation and THP-1 cell adhesion. Imidaprilat attenuated zinc/pyrithione-induced PKC activation and THP-1 cell adhesion. These data suggest that ACEI reduces MCP-1 or PMA-triggered monocyte adhesion to activated HUVECs by modulating labile zinc in monocytes. Our findings may point out a novel anti-inflammatory mechanism of ACEIs in atherogenesis.

Regulation of Gap Junction Intercellular Communication in Primary Canine Lens Epithelial Cells: Role of Protein Kinase C

Purpose: Gap junction intercellular communication (GJIC) is important in maintaining lens epithelial cell homeostasis and reductions in GJIC may be associated with the development of cataract. Protein kinase C (PKC) activation can disrupt gap junction communication via phosphorylation of connexin 43 (C×43) proteins that compose gap junction channels. This study examined the role of PKC activation in modulating GJIC in a primary canine lens epithelial cell (LEC) line. Methods: TPA (12-O-tetradecanoyl-phorbol-acetate), a potent PKC activator and inhibitor of GJIC, was utilized in the present study. Primary cultures of canine LEC were treated with TPA (0–1000 ng/ml) for 0.5 hr and GJIC was assessed by scrape loading/dye transfer (SL/DT), and immunoblotting to detect phosphorylation of C×43 protein. Inhibition of general and calcium-dependent PKC activity was achieved by pretreatment of cells with GF109203X and Gö6976, respectively. Results: Treatment with TPA (1–1000 ng/ml) significantly decreased GJIC in canine LEC as assessed by SL/DT. Pretreatment with 10 and 100 ng/ml TPA decreased GJIC by 80% as compared to controls and increased C×43 phosphorylation as assessed by immunoblotting. Pretreatment of cells with GF109203X and Gö6976, partially restored TPA-inhibited GJIC by 40% and 60%, respectively, and reduced C×43 phosphorylation. Expression of calcium dependent PKC isoforms was detected in canine whole lens and LEC. Conclusions: Treatment with TPA significantly reduces GJIC in canine LEC. These effects are mediated, in part, by activation of calcium-dependent PKC isoforms. Primary canine LEC are a useful model in the study of the molecular mechanisms involved in GJIC and cataractogenesis.

Role of ROS and MAPK in TPA‐induced ICAM‐1 expression in the myeloid ML‐1 cell line

Intercellular adhesion molecule 1 (ICAM-1) has been implicated in playing a key role in the mechanism of inflammatory process initiated in response to environmental agents, and during normal hematopoietic cell differentiation. Though induction of ICAM-1 by 12-O-tetradecanoyl-phorbol-13-acetate (TPA) in myeloid cells has been reported, the molecular mechanism by which TPA upregulates ICAM-1 expression remains unclear. In the present study, we investigated the signaling mechanism associated with TPA-induced ICAM-1 expression in ML-1 cells. Herein, our microarray, flow cytometry, and Western blot analysis indicated that ICAM-1 was constitutively expressed at a low level in ML-1 cells, but its expression was further upregulated at both the mRNA and protein levels in response to TPA. ICAM-1 expression in response to TPA was inhibited by pretreatment with GF109203X [a specific inhibitor of protein kinase C (PKC)], or with PD98059 and U0126 (specific inhibitors of MEK), suggesting the importance of PKC, and Erk1/2 signaling cascades in this response. Interestingly, ICAM-1 expression in response to TPA-induced PKC activation was linked to the generation of reactive oxygen species (ROS), as pretreatment with NAC (an ROS scavenger) blocked both ErK1/2 activation and ICAM-1 expression induced by TPA. In addition, TPA-induced ICAM-1 expression was blocked by inhibition of nuclear factor-kappaB (NF-kappaB) activation following pretreatment with BAY11-7085 (a specific inhibitor of NF-kappaB activation). TPA-induced NF-kappaB activation was shown by increased degradation of IkB (NF-kappaB specific inhibitory protein). Together, these observations demonstrated that TPA, a potent activator of PKC, induces ICAM-1 expression via a ROS- and ERK1/2-dependent signaling mechanism in ML-1 cells.

Protein kinase C shifts the voltage dependence of KCNQ/M channels expressed in Xenopus oocytes

It is well established that stimulation of G(q)-coupled receptors such as the M1 muscarinic acetylcholine receptor inhibits KCNQ/M currents. While it is generally accepted that this muscarinic inhibition is mainly caused by the breakdown of PIP(2), the role of the subsequent activation of protein kinase C (PKC) is not well understood. By reconstituting M currents in Xenopus oocytes, we observed that stimulation of coexpressed M1 receptors with 10 microm oxotremorine M (oxo-M) induces a positive shift (4-30 mV, depending on which KCNQ channels are expressed) in the conductance-voltage relationship (G-V) of KCNQ channels. When we applied phorbol 12-myristate 13-acetate (PMA), a potent PKC activator, we observed a large positive shift (17.8 +/- 1.6 mV) in the G-V curve for KCNQ2, while chelerythrine, a PKC inhibitor, attenuated the shift caused by the stimulation of M1 receptors. By contrast, reducing PIP(2) had little effect on the G-V curve for KCNQ2 channels; although pretreating cells with 10 mum wortmannin for 30 min reduced KCNQ2 current amplitude by 80%, the G-V curve was shifted only slightly (5 mV). Apparently, the shift induced by muscarinic stimulation in Xenopus oocytes was mainly caused by PKC activation. When KCNQ2/3 channels were expressed in HEK 293T cells, the G-V curve seemed already to be shifted in a positive direction, even before activation of PKC, and PMA failed to shift the curve any further. That alkaline phosphatase in the patch pipette shifted the G-V curve in a negative direction suggests KCNQ2/3 channels are constitutively phosphorylated in HEK 293T cells.

Opposing effects of phorbol‐12‐myristate‐13‐acetate, an activator of protein kinase C, on the signaling of structurally related human dopamine D1 and D5 receptors

The 'cross-talk' between different types of neurotransmitters through second messenger pathways represents a major regulatory mechanism in neuronal function. We investigated the effects of activation of protein kinase C (PKC) on cAMP-dependent signaling by structurally related human D1-like dopaminergic receptors. Human embryonic kidney 293 (HEK293) cells expressing D1 or D5 receptors were pretreated with phorbol-12-myristate-13-acetate (PMA), a potent activator of PKC, followed by analysis of dopamine-mediated receptor activation using whole cell cAMP assays. Unpredictably, PKC activation had completely opposite effects on D1 and D5 receptor signaling. PMA dramatically augmented agonist-evoked D1 receptor signaling, whereas constitutive and dopamine-mediated D5 receptor activation were rapidly blunted. RT-PCR and immunoblotting analyses showed that phorbol ester-regulated PKC isozymes (conventional: alpha, betaI, betaII, gamma; novel: delta, epsilon, eta, theta) and protein kinase D (PKCmicro) are expressed in HEK293 cells. PMA appears to mediate these contrasting effects through the activation of Ca2+-independent novel PKC isoforms as revealed by specific inhibitors, bisindolylmaleimide I, Gö6976, and Gö6983. The finding that cross-talk between PKC and cAMP pathways can produce such opposite outcomes following the activation of structurally similar D1-like receptor subtypes is novel and further strengthens the view that D1 and D5 receptors serve distinct functions in the mammalian nervous and endocrine systems.

Facile Synthesis and Radioiodine Labeling of Hypericin.

AbstractFor Abstract see ChemInform Abstract in Full Text.

Regulation of GLUT1-mediated glucose uptake by PKClambda-PKCbeta(II) interactions in 3T3-L1 adipocytes.

Members of the PKC (protein kinase C) superfamily play key regulatory roles in glucose transport. How the different PKC isotypes are involved in the regulation of glucose transport is still poorly defined. PMA is a potent activator of conventional and novel PKCs and PMA increases the rate of glucose uptake in many different cell systems. In the present study, we show that PMA treatment increases glucose uptake in 3T3-L1 adipocytes by two mechanisms: a mitogen-activated protein kinase kinase-dependent increase in GLUT1 (glucose transporter 1) expression levels and a PKClambda-dependent translocation of GLUT1 towards the plasma membrane. Intriguingly, PKClambda co-immunoprecipitated with PKCbeta(II) and did not with PKCbeta(I). Previously, we have described that down-regulation of PKCbeta(II) protein levels or inhibiting PKCbeta(II) by means of the myristoylated PKCbetaC2-4 peptide inhibitor induced GLUT1 translocation towards the plasma membrane in 3T3-L1 adipocytes. Combined with the present findings, these results suggest that the liberation of PKClambda from PKCbeta(II) is an important factor in the regulation of GLUT1 distribution in 3T3-L1 adipocytes.